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Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Jatrophane Diterpenoids from Euphorbia glomerulans Aobulikasimu Hasan,†,‡ Ge-Yu Liu,† Rui Hu,†,‡ and H. A. Aisa*,† †

The Key Laboratory of Plant Resources and Chemistry of Arid Zone and State Key Laboratory Basis of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi 830011, People’s Republic of China ‡ University of Chinese Academy of Sciences, Beijing 100039, People’s Republic of China

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ABSTRACT: In a phytochemical investigation of the whole plant of Euphorbia glomerulans, 17 new (1−17) and five known jatrophane diterpenoids (18−22) were identified. The X-ray crystallographic data of compounds 1, 4, and 21 permitted the definition of the absolute configurations of these compounds. The cytotoxicity and multidrug resistance reversal activities of the 17 new compounds were evaluated on multidrug-resistant MCF-7/ADR cells overexpressing P-glycoprotein. Several compounds showed different chemoreversal activities and considerably decreased cytotoxicity. Compounds 11 (IC50 value of 5.0 ± 0.8 μM) and 12 (IC50 value of 5.2 ± 2.0 μM) possessed MDR reversal activities that were as good as that of verapamil (IC50 value of 4.7 ± 0.6 μM).



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RESULTS AND DISCUSSION The molecular formula of euphoglomeruphane A (1) was deduced as C35H42O12 from its positive HRESIMS ([M + Na]+, m/z 677.2523, calcd for C35H42O12Na, 677.2568) and 13 C NMR data (Tables 1 and 2). The spectroscopic data of 1 revealed that it was structurally similar to 21, possessing an acetoxy group instead of a hydroxy group at C-15 in 21. Inspection of the 1H−1H COSY spectrum (key correlations are shown in bold in Figure 2A) permitted the identification of four fragments, which were connected according to HMBC correlations (Figure 2A). The correlations of H-1α and H-1β with C-4, C-14 (δC 204.1), and C-15 (δC 91.2); of H-4 (δH 2.82) with C-14 and C-15; of H-13 (δH 3.50) with C-12 (δC 133.4) and C-14; and of H3-20 with C-13 and C-14 confirmed the connection between fragment A (H-11/H-12, H-12/H-13, H-13/H3-20) and fragment B (H2-1/H-2, H-2/H-3, H-3/H-4, H-4/H-5, H-2/H3-16), which also placed an acetoxy group and a carbonyl at C-15 and C-14, respectively. Based on the cross-peaks from H-5 and H-7 to C-5, C-6, and C-7, fragment C (H-6/H-17, H-6/H-7, H-7/H-8) was attached to B. The correlations of H3-18 and H3-19 with C-9 (δC 205.2), C-10, and C-11 and of H-8 with C-9 (δC 205.2) and C-10 permitted establishing the molecular framework of compound 1. The correlations of oxymethine H-5 (δH 5.53), H-7 (δH 5.84), and H-8 (δH 5.34) with related carbonyl carbons (δC 168.6, 170.5, and 170.0) located the acetoxy moieties at C-5, C-7, and C-8, respectively. A benzoyloxy moiety was located at C-3 according to the cross-peaks of its carbonyl carbon (δC 165.6) with H-3

atrophane diterpenoids occur solely in the plants of the Euphorbiaceae family, and they have attracted considerable interest in new drug research and development because of their structural diversity and distinctive bioactivities.1−5 These macrocyclic diterpenoids possess a bicyclo[10.3.0]pentadecane skeleton without the presence of a cyclopropane ring, and their structures vary based on the position, nature, and number of double bonds and oxygen functionalities (epoxy, ether, polyester, carbonyl, and hydroxy groups) and the configuration of the diterpenoid core.6,7 Jatrophane diterpenoids have been studied for decades in the search for new drugs8 that reverse multidrug resistance (MDR) in pathogenic yeasts, viruses, and cancer cells.9 One of the reasons for most common tumors10 and pathogenic yeasts11−13 being resistant to the available drugs is MDR,14 which can evolve by various biochemical mechanisms, including increasing drug efflux or decreasing drug uptake.15 An important process related to MDR is the overexpression of P-glycoprotein (P-gp) (an efflux pump), which can reduce drug retention in cells.16,17 Many naturally occurring jatrophane derivatives18 and structurally modified compounds have shown higher potencies than cyclosporin A or verapamil; hence, they are promising P-gp inhibitors for further drug research.19 To find efficient reversal modulators from the Euphorbiaceae (spurge) family,20−23 the bioactive constituents of Euphorbia glomerulans were investigated. E. glomerulans Prokh. is distributed in northwestern China and the five countries that make up Central Asia. As part of a continuing study of new bioactive compounds from the genus Euphorbia, 17 new (1−17) and five known jatrophane diterpenoids (18−22) were identified from the whole plant of E. glomerulans. © XXXX American Chemical Society and American Society of Pharmacognosy

Received: July 7, 2018

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DOI: 10.1021/acs.jnatprod.8b00507 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Table 1. 1H NMR Spectroscopic Data (δ in ppm and J in Hz) for 1−5 position 1 2 3 4 5 7 8 9 11 12 13 14 16 17 18 19 20 2-OAc 3-OAc 5-OAc 7-OAc 8-OAc 9-OAc 14-OAc 15-OAc OBz 2, 6 3, 5 4 OH

1

2

3

4

5

δH, mult (Hz)

δH, mult (Hz)

δH, mult (Hz)

δH, mult (Hz)

δH, mult (Hz)

α 2.99 dd (15.7, 8.3) β 2.22 m 2.44 m 5.80 t (3) 2.82 dd (7.0, 3.0) 5.53 d (6.8) 5.84 s 5.34 s

α 2.91 dd (9.4, 6.1) β 2.11 m 2.39 m 5.71 t (3.7) 2.93 d (2.0) 5.92 s 5.58 s 4.65 d (9.6)

α 2.99 dd (15.7, 8.2) β 2.20 m 2.44 m 5.81 m 2.84 dd (8.2, 3.3) 5.68 d (8.1) 5.61 s 4.63 d (9)

5.93 m 5.91 m 3.50 m

5.86 s 5.91 dd (16.2, 9.1) 3.45 m

5.90 d (16.1) 5.82 m 3.49 m

α 2.90 dd (15.7, 9.1) β 2.25 m 2.62 m 5.84 m 3.30 dd (5.9, 3.2) 5.60 d (7.0) 6.04 s 5.18 d (10.1) 4.94 s 5.87 d (16.1) 5.62 d (9.4) 3.60 m

1.04 5.41 5.39 1.19 1.31 1.41

1.00 6.05 5.72 1.17 1.25 1.41

1.04 5.79 5.52 1.20 1.25 1.41

1.05 5.20 5.20 0.91 1.28 1.34

d (6.6) s s s s d (6.6)

d (6.5) s s s s d (6.5)

d (6.6) s s s s d (6.6)

d (6.7) s s s s d (6.6)

α 2.79 dd (15.4) β 2.17 dd (15.5) 5.76 3.18 5.01 5.83 5.79

s dd (5.2, 2.5) s s s

5.89 5.77 2.65 5.12 1.49 5.20 4.97 1.18 1.26 1.20 2.10

d (16.1) m m s s s s s s d (7.1) s

1.92 1.65 s 2.03 s 2.07 s

1.22 s

1.69 s 1.99 s

1.25 s 2.09 s 1.51 s

2.13 s 2.04 s 2.22 s

2.17 −3 8.00 7.43 7.56

s d (7.7) t (7.7) t (7.4)

2.14 −5 7.87 7.37 7.50 −8 3.13

s d (8.3) t (7.7) t (10.8, 4.0) d (9.7)

2.19 −3 7.99 7.43 7.56 −8 3.19

s d (7.1) t (7.7) t (7.4) d (9.3)

2.20 s −3/7 7.99 d (7.2)/8.08 d (7.2) 7.39 t (7.7)/7.45 t (7.7) 7.53 t (7.4)/7.57 t (7.4)

−3 8.02 d (7.2) 7.43 t (7.6) 7.56 t (7.4) −5/15 3.46 s

Finally, the correlation of C-15 (δC 90.5) with AcO-15 (δH 2.14) supported the attachment of an acetoxy moiety at C-15. Euphoglomeruphane C (3) was deduced to have a formula of C33H40O11 based on its HRESIMS ion at m/z 635.2463 [M + Na]+ (calcd for 635.2463). Comparison of the spectroscopic data of 3 with those of 21 suggested similar structures but with different esterification patterns. The HMBC correlations of HO-8 (δH 3.19)/C-8 (δC 71.6) and HO-8 (δH 3.19)/C-9 (δC 211.3) placed a hydroxy group at C-8. The HMBC cross-peaks between AcO-15 (δH 2.19) and C-15 (δC 91.0) placed the remaining acetoxy moiety at C-15. The molecular formula of euphoglomeruphane D (4) was assigned as C42H48O13 by the (+)-HRESIMS ion at m/z 783.2989 [M + Na]+ (calcd for 783.2987) and 13C NMR data. The NMR data indicated that the structure of 4 was similar to 20. The difference between these two jatrophane diterpenoids was the presence of a benzoyloxy group at C-7 in 4 rather than the acetoxy group in 20, which was determined by the crosspeak between H-7 and the carbonyl carbon of the benzoyloxy group. H-4 was assigned as α orientation based on biogenetic considerations.30 The NOESY interactions of H-2 with H-4, H-2 with H-3, and H-4 with H-13 demonstrated that the two methyl groups at C-2, C-13, and 3-OBz were β-oriented. The correlations of H-5 with H-8 and AcO-15 and the absence of a correlation between H-5 and H-7 confirmed the β orientation

(δH 5.80) and BzO-3 (H-2′, H-3′) (δH 8.00, 7.43). Finally, the correlation between AcO-15 (δH 2.17) and C-15 (δC 91.2) showed the presence of an acetoxy group at C-15. The NOEs (Figure 2B) of H-3/H-4α, H-4α/H-13, H-4α/H-7, H-5β/H-8, and AcO-15/H-5β and a comparison with reported compounds supported the structural assignment of euphoglomeruphane A (1) (Figure 2). The large coupling constant (7.0 Hz) between H-4 and H-5 revealed that compound 1 is in an endotype conformation.21,23 In addition, X-ray crystal diffraction data of 1 confirmed that compound 1 is an endotype conformer. The absolute configuration of euphoglomeruphane A (1) was assigned as (2S,3S,4S,5R,7S,8R,13S,15R)-5,7,8,15-tetraacetoxy-3-benzoyloxy-9,14-dioxojatropha-6(17),11E-diene. The (+)-HRESIMS data of euphoglomeruphane B (2) showed an [M + Na]+ ion at m/z 635.2463 (calcd for 635.2463), corresponding to the molecular formula C33H40O11. A comparison of the 13C NMR data (Table 2) of 2 and 21 showed that the two compounds are structurally related, but differing with respect to the esterification pattern. The HMBC correlations of the carbonyl carbons (δC 169.9, 164.8) and oxymethine protons (δH 5.71, 5.92) placed the acetoxy group at C-3 and the benzoyloxy group at C-5. The HMBC correlations of HO-8 (δH 3.13)/C-8 (δC 71.6) and HO-8 (δH 3.13)/C-9 (δC 211.5) placed a hydroxy moiety at C-8. B

DOI: 10.1021/acs.jnatprod.8b00507 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products Table 2.

13

position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2-OAc 3-OAc 5-OAc 7-OAc 8-OAc 9-OAc 14-OAc 15-OAc OBz CO 1 2, 6 3, 5 4

Article

C NMR Spectroscopic Data (δ in ppm and J in Hz) for 1−6 1

2

3

4

5

6

δC

δC

δC

δC

δC

δC

44.7 39.2 76.7 50.3 70.6 137.8 65.0 73.2 205.2 49.6 135.2 133.4 43.8 204.1 91.2 14.1 120.5 23.9 25.9 20.3

45.1 39.1 77.0 49.8 73.5 137.0 64.6 71.6 211.5 48.5 134.5 134.3 43.7 204.0 90.5 14.0 125.7 25.4 23.5 20.4

45.0 39.2 77.0 50.3 71.3 137.8 66.0 71.6 211.4 48.7 134.6 133.6 43.7 204.2 91.0 14.1 122.9 25.5 23.4 20.4

44.1 38.4 77.5 50.2 70.5 140.3 67.7 70.4 81.2 40.4 135.8 130.8 44.8 204.6 92.1 14.6 119.1 25.8 23.9 20.5

168.6, 20.8 170.4, 21.0

168.8, 20.4

49.9 88.8 80.5 45.6 70.3 143.7 67.1 70.8 205.2 50.3 135.0 134.6 37.8 81.4 84.7 21.4 111.4 24.3 25.4 23.4 170.3, 22.3

53.3 79.2 82.9 45.8 70.4 143.7 68.0 71.3 205.2 50.2 135.2 135.1 36.5 82.4 84.8 23.9 111.5 24.7 25.8 23.3

170.1, 20.9 170.7, 20.7

170.6, 20.7

171.6, 21.1

172.2, 21.2

3− 165.2 129.9 130.0 128.9 133.6

3/7− 165.5/165.9 130.1/129.3 130.0/130.3 128.8/128.7 133.4/133.8

169.9, 20.7 168.6, 20.7 170.5, 20.9 170.0, 20.6

170.7, 20.2

169.3, 21.4 3− 165.5 130.1 129.8 128.7 133.5

169.4, 21.5 5− 164.8 129.7 129.9 128.6 133.5

170.3, 21.1 170.2, 20.5 169.4, 21.4 3− 165.7 130.1 129.7 128.7 133.5

169.7, 21.7 3/7− 165.6/165.8 130.3/130.3 129.8/129.9 128.5/129.0 133.3/133.8

diterpenoid and structurally related to 5. Compared to 5, a hydroxy and a benzoyloxy group were located at C-2 and C-7, respectively. The NOEs of H-4/H-3, H-7/H-4, and H-4/H-13 revealed their cis (conventionally α) orientation. In addition, the NOESY interactions of H-1β with H3-16, H-5 with H-8, H14 with H3-20, and H-8 with HO-15 indicated the β orientation of the 2-methyl group and 15-OH. Euphoglomeruphane G (7), isolated as a white amorphous powder, possessed a molecular formula of C33H40O10 assigned by the (+)-HRESIMS ion at m/z 619.2495 [M + Na]+ (calcd for 619.2514). The NMR data indicated that 7 had a jatrophane skeleton and was structurally similar to 22, except for the presence of acetoxy groups at C-3 and C-15 and a benzoyloxy moiety at C-5 in 7 rather than the BzO-3, AcO-5, and HO-15 moieties in 22. Based on the correlations of H-3 (δH 5.74)/carbonyl carbon (δC 169.9), H-1α (δH 2.93)/C-3 (δC 77.02), and H3-16 (δH 1.01)/C-3 (δC 77.02), an acetoxy group was located at C-3. The correlations of H-5 (δH 5.93)/ (δC 165.0), H-5 (δH 5.93)/C-7 (δC 64,5), and H-5 (δH 5.93)/ C-17 (δC 122.6) placed a benzoyloxy group at C-5. The HMBC correlation between AcO-15 (δH 2.12) and C-15 (δC 90.6) located the remaining acetoxy group at C-15. The NOEs of H-2/H-4, H-3/H-4, H-4/H-13, and H-4/H-7 indicated that the C-2 and C-13 methyl groups and the acetoxy groups at C-3

of 7-OBz and 15-OAc, while the cross-peaks among H-5, H-8, and H-9 suggested the α orientation of the acetoxy groups at C-5, C-8, and C-9. The absolute configuration of euphoglomeruphane D (4) was confirmed by X-ray crystallographic analysis and named (2S,3S,4S,5R,7S,8S,9S,13S,15R)-5,8,9,15tetraacetoxy-3,7-dibenzoyloxy-14-oxojatropha-6(17),11Ediene. The (+)-HRESIMS data of euphoglomeruphane E (5) displayed a sodium adduct ion [M + Na]+ at m/z 695.2635 (calcd for 695.2674), corresponding to the molecular formula C35H44O13. The NMR data (Tables 1 and 2) revealed that 5 was a jatrophane-type diterpenoid and structurally similar to 21, differing only in their substituents. Compared to 21, compound 5 had a hydroxy group at C-5 and acetoxy groups at C-2 and C-14. The NOESY correlations between H-1β and H3-16, H-3 and H-4, H-4 and H-13, and H-4 and H-7 suggested β orientation of the methyl groups at C-2 and C-13, 3-OBz, and 7-OAc, while the correlations of H-5/H-8, H-14/ H3-20, and HO-15/H-5 showed that these protons were βoriented. The molecular formula of euphoglomeruphane F (6) was determined as C38H44O12 according to its (+)-HRESIMS ion at m/z 715.2681 [M + Na]+ (calcd for 715.2725). Analysis of the NMR data revealed that 6 was also a jatrophane-type C

DOI: 10.1021/acs.jnatprod.8b00507 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 1. Structures of euphoglomeruphane A−V (1−22) isolated from Euphorbia glomerulans.

C-15, and C-7, respectively, instead of acetoxy groups in 1. The above results were confirmed by the shielded chemical shifts of C-2 (δC 79.0), C-7 (δC 64.8), H-7 (δH 6.30), and C-15 (δC 85.5) as well as the HMBC correlations of the benzoyloxy carbonyl carbon (δC166.2), C-6, and C-9 with H-7 (δH 6.30) and of HO-15 (δH 4.57) with C-15 (δC 85.5). The NOESY correlations of 9 indicated that it had the same relative configuration as 1. The molecular formula of euphoglomeruphane J (10) was determined as C38H42O11 based on the (+)-HRESIMS (m/z 697.2607 [M + Na]+, calculated for 697.2619) and 13C NMR data. Comparison of the NMR data of 10 with those of 3 revealed that the compounds were highly similar, except for the linkage of a benzoyloxy group at C-7 instead of the 7-OAc in 3. It was confirmed by the deshielded chemical shift of H-7 (δH 5.91) and the HMBC correlation from H-7 (δH 5.91) to the carbonyl carbon (δC 165.7). The NOESY correlations of 10 indicated that it had the same relative configuration as 3. The (+)-HRESIMS data of euphoglomeruphane K (11) showed an [M + Na]+ ion at m/z 679.2710 (calculated for 679.2725), corresponding to the molecular formula C35H44O12. Analysis of the NMR data revealed that compound 11 was a jatrophane-type diterpenoid with two acetoxy groups, two hydroxy groups, a benzoyloxy group, and an isobutanoyloxy group. The locations of these substituents were confirmed through the HMBC correlations between the carbonyl carbons and corresponding oxymethine protons. The HMBC crosspeaks from HO-15 (δH 4.30) to C-4 (δC 47.9), C-1 (52.4), C14 (213.4), and C-15 (85.3) revealed that a hydroxy group was located at C-15. Based on the HMBC correlations from H-3 (δH 5.54) to the carbonyl carbon (δC 165.8), a benzoyloxy moiety was placed at C-3. According to the HMBC cross-peaks from H-8 (δH 5.33) to C-9 (δC 204.9) and the carbonyl carbon (δC 176.1), an isobutanoyloxy moiety was located at C-8. An acetoxy group was located at C-7 based on the correlations from H-7 (δH 5.96) to C-6 (δC138.5), C-9 (204.9), and the

Figure 2. Key HMBC, 1H−1H COSY, and NOESY correlations of euphoglomeruphane A (1).

and C-7 were on the β face of the macrocycle. Subsequently, the lack of a NOE between H-5 and H-7 confirmed that H-5 was β-oriented. The chemical shifts and coupling constants of 7 were similar to those of 22. These observations permitted the definition of the structure of 7. The 13C NMR data of euphoglomeruphane H (8), with a molecular formula of C33H40O10 by an HRESIMS ion at m/z 619.2500 [M + Na]+ (calculated for 619.2514), were highly similar to those of 22, except for the presence of an acetoxy group at C-15 instead of the HO-15 in 22. An acetoxy group was attached to C-15 based on the HMBC correlation between AcO-15 (δH 2.19) and C-15 (δC 91.1). The NOESY correlations of H-4/H-3, H-4/H-13, and H-4/H-7 indicated that these protons were on the α face of the macrocycle, while the lack of correlation between H-5 and H-7 suggested that H5 was β-oriented. The correlation between H-5 and AcO-15 indicated the β orientation of AcO-15. Euphoglomeruphane I (9) possessed a molecular formula of C38H42O12 as deduced from its (+)-HRESIMS (m/z 713.2548 [M + Na]+, calculated for 713.2568) and NMR data. Analysis of 13C NMR data indicated that 9 was a jatrophane-type diterpenoid and structurally similar to 1, but differing in the presence of hydroxy moieties and a benzoyloxy group at C-2, D

DOI: 10.1021/acs.jnatprod.8b00507 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 3. ORTEP diagram of compounds 1, 4, and 21.

Thus, according to the cross-peaks from H-7 (δH 5.94) and H8 (δH 5.29) to the carbonyl carbons at δC 170.5 and 176.1, respectively, an acetoxy and an isobutanoyloxy moiety were confirmed at C-7 and C-8, respectively. The cross-peaks of HO-15 (δH 4.29) with C-4 (δC 48.0), C-1 (δC 49.9), and C-14 (δC 212.3) revealed that a hydroxy group was located at C-15. The NOESY spectrum of 12 indicated that it had the same relative configuration as 11. Euphoglomeruphane M (13), isolated as a white powder, had a molecular formula of C37H46O13 based on its 13C NMR and (+)-HRESIMS (m/z 721.2811 [M + Na]+, calculated for 721.2830) data. Its highly similar coupling constants and NMR chemical shifts indicated that 13 was an isomer of 12, differing in the substituents at C-7 and C-8. However, the absence of certain key HMBC correlations did not allow the locations of the acetoxy and isobutanoyloxy groups at C-7 and C-8 to be differentiated. However, the chemical shifts of H-7 (δH 5.93), H-8 (5.34), C-7 (δC 65.0), and C-8 (73.1) and the molecular structure were quite similar to those of 12 and 5α,8αdiacetoxy-15β-hydroxy-3β-benzoyloxy-7β-isobutanoyloxyjatropha-6(17),11E-diene-9,14-dione25 (δH‑7 5.92, δH‑8 5.35, δC‑7 65.1, and δC‑8 73.3), indicating that variations in the ester groups between isomers (jatrophane skeleton) have little effect on the chemical shifts of their attached carbons. Therefore, an isobutanoyloxy and an acetoxy moiety were placed at C-7 and C-8, respectively, based on the HMBC cross-peaks from H-7 (δH 5.93) to the carbonyl carbon (δC 176.5) of the isobutanoyloxy group and from H-8 (δH 5.34) to the carbonyl carbon (δC 170.1) of the acetoxy group. Thus, the structure of 13 was defined as shown.

carbonyl carbon (170.6). Compared to those in 12 and 13, the resonance of C-2 in 11 was shielded (ΔδC −9.1), indicating the presence of a hydroxy moiety. Assuming that H-4 was αoriented, the NOEs indicated that HO-2, H-3, H-7, H-13, and H3-18 were also α-oriented, while H-5, H-8, HO-15, H3-16, H3-19, and H3-20 were in the β position. These interpretations allowed the structure of compound 11 to be established. The molecular formula (C37H46O13) of euphoglomeruphane L (12) was deduced from the (+)-HRESIMS (m/z 721.2814 [M + Na]+, calcd 721.2830) and 13C NMR data. The 1H and 13 C NMR data of 12 showed high similarities to 11, except for the presence of an acetoxy group at C-2 instead of the hydroxy moiety in 11. The deshielded chemical shift of C-2 (ΔδC +9.1) suggested the presence of an acetoxy moiety in 12. The protons at δH 5.94 and 5.29 were tentatively deduced as H-7/ H-8 or H-8/H-7. However, HMBC correlations from these two protons to nearby carbons, such as C-5, C-6, and C-9, were not observed; therefore, sufficient experimental evidence was not available for their assignment. This phenomenon, namely, the absence of HMBC correlations from H-7 and H-8 to nearby carbons, has been previously reported.24 However, the structure and chemical shifts of H-7 (δH 5.94), H-8 (5.29), C-7 (δC65.6), and C-8 (72.5) were highly similar to those of 11, 13, and 5α,8α-diacetoxy-15β-hydroxy-3β-benzoyloxy-7βisobutanoyloxyjatropha-6(17),11E-diene-9,14-dione25 (δH‑7 5.92, δH‑8 5.35, δC‑7 65.1, and δC‑8 73.3). By comparing the NMR data to those of the above-mentioned compounds, the proton at δ H 5.94 was determined to be H-7, and consequently, the proton at δH 5.29 was identified as H-8. E

DOI: 10.1021/acs.jnatprod.8b00507 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 3. 1H NMR Spectroscopic Data (δ in ppm and J in Hz) for 6−10 position 1 2 3 4 5 7 8 11 12 13 14 16 17 18 19 20 3-OAc 5-OAc 7-OAc 8-OAc 14-OAc 15-OAc OBz 2, 6 3, 5 4 OH

6

7

8

9a

10

δH, mult (Hz)

δH, mult (Hz)

δH, mult (Hz)

δH, mult (Hz)

δH, mult (Hz)

α 2.93 m β 2.10 m 2.39 m 5.74 t (3.5) 2.95 m 5.93 s 5.63 s α 2.92 m β 2.23 t (11.2) 5.91 s 5.76 dd (16) 3.44 m

α 2.99 dd (15.8, 8.4) β 2.20 m 2.44 m 5.86 t (3.4) 2.82 dd (8.0, 3.3) 5.66 d (8.0) 5.59 d (9.7) α 2.95 dd (12.3, 1.7) β 2.24 m 5.85 d (16.0) 5.78 dd (16.0, 9.2) 3.47 m

1.01 5.62 5.40 1.14 1.18 1.40 1.93

1.04 5.44 5.32 1.17 1.18 1.40

α 2.25 m β 2.26 m 5.49 3.42 5.05 6.07 5.88

d (3.5) brs s s s

5.90 5.84 3.11 5.19 1.32 5.27 5.13 1.14 1.29 1.22

d (16.0) dd (16.0, 9.3) m s s s s s s d (7.0)

d (6.6) s s s s d (6.6) s

1.24 s

d (6.6) s s s s d (6.6)

1.69 s 1.98 s

5.59 3.69 5.77 6.30 5.52

d (3.8) brd (7.0) brs s m

α 3.03 dd (15.9, 8.3) β 2.23 m 2.50 m 5.84 t (3.6) 2.98 dd (8.7, 3.1) 5.74 d (9.2) 5.91 brs 4.77 d (9.1)

6.14 d (16.0) 5.89 d (16.0, 10.1) 4.64 m

6.04 d (16.0) 5.87 m 3.55 m

1.35 5.63 5.51 1,25 1.33 1.39

1.06 5.95 5.62 1.24 1.31 1.47

s brs s s s d (6.5)

1.17 s

2.12 s 2.24 s −3/7 8.00 d (8.1)/8.10 d (8.1) 7.41 t (7.6)/7.46 t (7.6) 7.54 t (7.4)/7.58 t (7.4) −2/5/15 2.03 s/3.97 s/3.45 brs

α 2.59 d (15.3) β 2.27 d (15.3)

s s s s s d (6.5)

1.25 s

2.10 s 2.12 −5 7.90 7.38 7.51

s d (8.3) t (7.7) t (7.4)

2.19 −3 7.99 7.43 7.56

s d (7.1) t (7.8) t (7.4)

−3/7 8.00 d (7.3)/8.08 br s 7.53 t (7.9)/7.51 t (7.7) 7.64 t (7.4)/7.67 t (7.4) −2/15 4.81 s/4.57 s

2.24 s −3/7 7.97 d (7.7)/7.97 d (7.7) 7.43 t (7.5)/7.42 t (7.5) 7.56 m/7.56 m −8 3.33 s

a

Data were recorded in acetone-d6 at 800 MHz (1H) and 200 MHz (13C).

(+)-HRESIMS (m/z 677.2549, calculated for 677.2568) data. Interpretation of its NMR data revealed the structure of 17 to be similar to 1, except for the presence of a 3-oxobutanoyloxy, BzO-7, and HO-15 groups, instead of BzO-3, AcO-7, and AcO15 groups in 1, respectively. The relative configuration of 17 was the same as that of 1. The five known jatrophane diterpenoids were identified as 3β,7β,8α,15β-tetraacetoxy-5α-benzoyloxyjatropha-6(17),11Edien-9,14-dione (18),26 3β,7β,8α,9α,15β-pentaacetoxy-5αbenzoyloxyjatropha-6(17),11E-dien-14-one (19), 2 7 5α,7β,8α,9α,15β-pentaacetoxy-3β-benzoyloxyjatropha6(17),11E-dien-14-one (20),27 (2S,3S,4S,5R,7S,8R,13S,15R)5,7,8-triacetoxy-15-hydroxy-3-benzoyloxy-9,14-dioxojatropha6(17),11E-diene (21),27 and 5α,7β-diacetoxy-15β-hydroxy-3βbenzoyloxyjatropha-6(17),11E-diene-9,14-dione (22)25 by comparison of their NMR and MS data to the reported spectroscopic data. The MDR reversal abilities and cytotoxicity of the new jatrophane diterpenoids were evaluated in the MCF-7 cells and P-gp-overexpressing MCF-7/ADR cells by the MTT method. The results revealed that these compounds showed different chemoreversal activities and much lower cytotoxicities. In particular, compounds 11 and 12 possessed MDR reversal activities with RF (reversal fold) values of 12.9 and 12.3 at 10 μM, respectively, which was as good as that of the positive control, verapamil (RF = 13.7).

The NMR data of euphoglomeruphane N (14), which had a molecular formula of C31H38O10 deduced by its (+)-HRESIMS data (m/z 593.2345 [M + Na]+, calcd 593.2357), were quite similar to those of 3, except for the presence of a hydroxy moiety at C-15 in 14 instead of an acetoxy group in 3. This structural assignment was supported by the correlation of HO15 (δH 4.21) with the acetyl carbonyl carbon (δC 212.6). Euphoglomeruphane O (15) had a molecular formula of C40H44O12 as assigned by its 13C NMR (Table 6) and (+)-HRESIMS data (m/z 739.2706 [M + Na]+, calculated for 739.2725). Its 1H and 13C NMR data were highly similar to those of 2. The only difference involved the presence of a benzoyloxy group at C-8 in 15, which was replaced by a hydroxy group in 2. This was defined by the correlation of H-8 (δH 6.16) with the carbonyl carbon (δC 166.2). The (+)-HRESIMS data of euphoglomeruphane P (16) showed an ion at m/z 739.2700 [M + Na]+ (calcd 739.2725), which corresponded to a molecular formula of C40H44O12. Comparison of its NMR data with those of 1 revealed that they were highly similar, except for the presence of a benzoyloxy moiety at C-7 in 16 rather than an acetoxy group in 1. This was confirmed by the HMBC cross-peak of the carbonyl carbon of the benzoyloxy (δC 166.1) group with H-7 (δH 6.12). Euphoglomeruphane Q (17) had a molecular formula of C35H42O12 according to its NMR (Tables 6 and 7) and F

DOI: 10.1021/acs.jnatprod.8b00507 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products Table 4.

13

position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2-OAc 3-OAc 5-OAc 7-OAc 8-OAc 15-OAc OBz CO 1 2, 6 3, 5 4 iBu CO 1 2 3

Article

C NMR Spectroscopic Data (δ in ppm and J in Hz) for 7−12 7

8

9

10

11

δC

δC

δC

δC

δC

45.1 39.1 77.0 49.9 72.9 139.2 64.5 41.1 210.1 50.7 135.7 133.1 43.7 204.2 90.7 14.0 122.6 24.5 23.5 20.5

44.9 39.2 77.1 50.5 70.8 140.3 65.8 41.1 209.6 50.8 135.7 132.9 43.9 204.4 91.1 14.1 120.7 24.2 23.4 20.3

12 δC

54.0 79.0 82.4 48.6 74.1 138.7 64.8 74.1 204.9 49.6 136.5 134.9 43.3 213.9 85.5 22.9 124.4 24.5 25.9 21.0

44.9 39.0 77.1 50.1 71.7 137.2 65.9 71.7 211.1 48.4 134.6 133.5 43.5 204.0 90.7 13.8 ND* 25.4 23.3 20.2

52.4 79.5 81.2 47.9 79.5 138.5 NDa 72.6 204.9 49.8 136.4 133.1 43.0 213.4 85.3 23.5 119.2 24.2 25.5 20.9

49.9 88.6 78.6 48.0 NDa NDa 65.6 72.5 204.6 49.9 136.7 132.4 44.0 212.3 85.3 19.2 110.2 24.1 25.3 20.8 169.0, 20.9

169.0, 20.4

168.6, 20.2

169.0, 20.9 170.6, 20.9

170.0, 22.5 170.5, 20.9

3− 165.8 129.8 130.2 128.7 133.6 8− 176.1 18.9 33.7 19.0

3− 165.4 129.6 130.3 128.8 133.7 8− 176.1 18.9 33.7 19.0

169.9, 20.7 170.7, 20.5

170.7, 20.2

169.3, 21.5 5− 165.0 129.6 129.9 128.6 133.5

168.8, 20.8 3− 165.6 130.1 129.8 128.7 133.5

170.3, 20.5 3/7− 166.2/165.9 130.8/130.5 130.5/130.4 134.5/134.1 129.4/129.5

169.2, 21.2 3/7− 165.6/165.7 129.0/129.0 129.8/129.5 128.5/128.4 133.2/133.6

a

ND: the signal was not detected.

all of the missing signals were detected, and previous structural assignments were confirmed. The 3D structure analysis showed that compounds 1−22 adopted an endotype conformation, which rarely occurs in naturally derived jatrophane diterpenoids. According to previous literature, the absence of expected signals could be caused by conformational exchange of the molecules or intramolecular chemical variations, inducing exchange of the nuclei between conformers and variations in the conformational equilibrium in the magnetic environment. Conformational investigations of jatrophane polyesters revealed that they could adopt exo- or endotype conformations. The particular combination of substituents could influence the conformer interconversion rates. In addition, their exchange rate was influenced by intermolecular interactions with the solvent,24 which is a challenge for separating jatrophane diterpenoids and worthy of further detailed studies.

Since compounds 1−3 and 7−17 possess a structurally homogeneous skeleton, differing only in the substitution pattern, an investigation on the structure−activity relationship (SAR) was possible. By comparing the substituents and reversal fold values of compounds 11, 12, and 13, we hypothesized that the presence of an isobutanoyloxy moiety at C-8 rather than at C-7 had a positive role in the modulation of drug accumulation in the MCF-7/ADR cells. In addition, a comparison of the biological evaluation results for compounds 1, 3, and 8 to those of 2, 7, and 15 suggested the following trend in activity for different substituents at C-8: benzoyloxy group > H ≈ hydroxy group. Interestingly, expected 13C NMR, HMBC, and HSQC correlations were missing or not detected for some isolates (9, 10, 11, 12, 13, 14, 15, and 21) (the missing signal area consisted of C-5, C-6, C-7, C-17). This observation indicated that some signals might be invisible because of their low intensities.24 As an additional study, compound 9 was evaluated at 800 MHz NMR (in acetone-d6). Consequently, G

DOI: 10.1021/acs.jnatprod.8b00507 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 5. 1H NMR Spectroscopic Data (δ in ppm and J in Hz) for 11−15 position 1 2 3 4 5 7 8 11 12 13 16 17 18 19 20 2-OAc 3-OAc 5-OAc 7-OAc 8-OAc 15-OAc OBz 2, 6 3, 5 4 OH



OiBu 1 2 3

11

12

13

14

15

δH, mult (Hz)

δH, mult (Hz)

δH, mult (Hz)

δH, mult (Hz)

δH, mult (Hz)

α 2.47 d (15.4) β 2.24 d (15.4)

α 3.16 d (16.2) β 2.27 d (16.4)

α 3.14 d (15.2) β 2.28 d (16.4)

5.54 3.55 5.45 5.96 5.33 5.99 5.80 4.37 1.39 5.60 5.33 1.23 1.36 1.37

5.90 3.27 5.42 5.94 5.29 5.94 5.80 3.75 1.60 5.42 5.31 1.20 1.34 1.35 1.66

5.92 3.25 5.42 5.93 5.34 5.97 5.79 3.77 1.60 5.42 5.32 1.20 1.31 1.35 1.55

d (3.5) dd (7.9, 3.8) brs s s d (16.0) dd (16.0, 10) m s brs s s s d (6.6)

d (3.5) brs brs m s m dd (16, 9.6) m s brs s s s m s

m brs brs s s d (16.0) dd (16.0, 9.6) m d (6.6) brs s s s d (6.6) s

α 2.46 d (14.4, 8.5) β 1.95 m 2.35 m 5.81 t (3.5) 2.84 dd (8.8, 3.1) 5.61 brs 5.65 s 4.60 d (9.5) 5.95 d (16.1) 5.71 dd (16.1, 9.7) 3.61 m 1.04 d (6.6) 5.84 brs 5.72 m 1,20 s 1.28 s 1.41 d (6.5)

α 2.94 dd (15.6, 8.4) β 2.11 m 2.44 m 5.72 t (3.7) 3.06 dd (9.6, 3.9) 5.92 d (12.5) 5.45 s 6.16 s 6.04 d (16.0) 5.87 m 3.55 m 1.01 d (6.6) 5.71 s 5.60 s 1.23 s 1.31 s 1.44 d (6.6) 1.89 s

1.58 s 2.04 s

2.17 s 2.02 s

2.18 s

1.71 s 1.98 s

2.10 s

2.04 s −3 8.13 d (7.6)) 7.46 t (7.8) 7.58 t (7.4) −2/15 1.92 brs/4.30 s −8 1.11 d (6.9) 2.62 m 1.16 d (7.0)

−3 8.12 7.45 7.56 −15 4.29 −8 1.09 2.59 1.13

d (7.4) t (7.3) t (6.9) s d (6.7) m d (6.8)

−3 8.13 7.45 7.57 −15 4.30 −7 1.13 2.51 1.16

d (7.2) t (7.7) t (7.4) s

−3 8.10 7.43 7.54 −15 4.21

d (7.4) t (7.7) t (7.4)

2.12 s −5/8 7.60 m/7.61 m 6.98 t (7.3)/7.08 t (7.6) 7.17 t (7.5) /7.28 t (7.3)

s

d (6.8) m d (7.1)

silica gel CC (petroleum ether−EtOAc gradient, from 65:1 to 0:1) to yield 16 fractions (Fr. 1−16). Fr. 11 (3.5 g) was fractionated by CC (Sephadex LH-20, 105 × 3 cm) to obtain seven subfractions (11A−11G). Fr. 11B was further purified by CC (silica gel, eluted with a gradient of CHCl3−acetone, from 100:1 to 0:1) to afford 10 fractions (11B1−11B10). Fractions 11B3, 11B4, 11B5, and 11B6 were further purified by semipreparative HPLC (CH3CN−H2O, 56:44, 55:45, 52:48, 49:51, respectively) to afford compounds 18 (8.1 mg), 15 (2.0 mg), 2 (27.7 mg), 3 (40.0 mg), and 14 (1.3 mg) as well as crystalline compounds 1 (30.0 mg) and 4 (7.2 mg). Fr. 12 (3.3 g) was fractionated by CC (Sephadex LH-20, 130 × 2.5 cm) to afford five fractions (12A−12E). Fraction 12BC was further separated using a Flash-ODS column (MeOH−H2O, 30−80%) to afford 10 subfractions (12BC1−12BC10), and compounds 5 (4.0 mg), 6 (2.0 mg), 17 (6.0 mg), 19 (2.4 mg), and 20 (5.5 mg) were obtained from fractions 12BC3 and 12BC5 by semipreparative HPLC (CH3CN−H2O, 50:50 and 49:51, respectively). Fr. 10, eluted with CHCl3−MeOH, 1:1, v/v, was separated using CC (Sephadex LH-20, 105 × 3 cm) to afford seven subfractions (10A−10G). Fraction 10BC, eluted with a CHCl3−acetone gradient from 100:1 to 0:1, was further chromatographed on silica gel to afford 13 subfractions (10BC1−10BC13). Fraction 10BC9 was fractionated by CC (Sephadex LH-20, 150 × 1.2 cm) to give six subfractions (10BC9A−10BC9F). Compounds 9 (13.0 mg), 10 (1.8 mg), and 11 (2.2 mg) were isolated by fractionation of 10BC9C using semipreparative HPLC (CH3OH−H2O, 63:37). Fractionation of 10BC6 was achieved by semipreparative HPLC (CH3CN−H2O, 47:53) and

EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were measured with a Büchi melting point B-450 apparatus. Optical rotation measurements were conducted on an Autopol VI automatic polarimeter in MeOH as the solvent. UV spectra in MeOH were acquired with a UV-2550 UV−visible spectrophotometer. IR data were recorded on a Nicolet 6700 spectrometer. 1D and 2D NMR spectra were acquired on Varian INOVA 400, 600, or 800 MHz NMR spectrometers in CDCl3 or CD3COCD3 as solvents. Positive ion HRESIMS data of the new jatrophane diterpenoids were acquired on a QSTAR Elite LC-MS/MS spectrometer. Semipreparative HPLC separations were conducted on a DIONEX UltiMate 3000 instrument equipped with an X Bridge RP-18 (5 μm, 10 × 150 mm) column. Silica gel column chromatography (CC) and Sephadex (LH-20) CC were used in the fractionation of extracts. Flash chromatography was carried out with a Combi Flash Rf System. Plant Material. E. glomerulans was collected in July 2016 from Manas, Xinjiang, China. The plant was identified by Prof. H. L. Feng (Xinjiang Institute of Ecology and Geography, CAS). A voucher specimen (XJBI-00025189) was deposited at Xinjiang Technical Institute of Physics and Chemistry, CAS. Extraction and Isolation. The air-dried whole plant of E. glomerulans (4.7 kg) was shredded and percolated with acetone at room temperature. The dried crude extract (358 g) was suspended in CH3CN and further partitioned with cyclohexane to give the CH3CNsoluble extract (105 g). The CH3CN-soluble extract was separated by H

DOI: 10.1021/acs.jnatprod.8b00507 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 6. 13C NMR Spectroscopic Data (δ in ppm and J in Hz) for 13−17 13 position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2-OAc 3-OAc 5-OAc

δC

δC

49.8 88.6 78.6 48.1 NDa ND 65.0 73.1 204.3 49.7 136.8 132.2 43.9 212.3 85.5 19.2 114.7 24.1 25.3 20.7 169.1, 20.8

46.7 39.0 77.9 51.0 73.3 138.2 65.7 71.4 211.0 48.7 135.8 132.7 44.0 212.6 84.9 14.1 ND 25.2 23.5 21.3

170.0, 22.5

168.9, 21.0 170.4, 21.0

15-OAc OBz CO 1 2, 6 3, 5 4 iBu CO 1 2 3 OAcAc CO

15 δC 45.0 39.2 77.0 49.7 73.2 137.2 74.0 64.4 204.8 49.5 135.5 133.8 43.8 204.2 90.7 14.1 ND 24.1 26.0 20.5

16 δC

17

16

δC

45.0 39.2 77.1 50.2 71.6 137.4 65.0 73.7 204.7 49.5 135.5 133.5 43.8 204.2 91.0 14.1 122.0 24.0 25.9 20.4

45.4 38.6 76.6 52.9 68.3 142.9 68.9 74.9 205.4 48.8 136.6 133.5 45.1 211.9 91.8 13.4 113.1 23.0 27.9 20.3

169.0, 20.4

169.6, 21.1

170.1, 20.7

170.1, 20.6

position 1 2 3 4 5 7 8 11 12 13 16 17α 17β 18 19 20 2-OAc 3-OAc 5-OAc 8-OAc 15-OAc OBz 2, 6 3, 5 4 OH

169.7, 20.8

7-OAc 8-OAc

14

Table 7. 13C NMR Spectroscopic Data (δ in ppm and J in Hz) for 16 and 17

170.1, 20.7

170.1, 20.6 3− 165.3 129.7 130.3 128.7 133.7 7− 176.5 18.6 33.9 19.5

3− 166.1 130.0 130.1 128.7 133.3

169.4, 21.5 5/8− 165.3/166.2 129.0/129.6 129.9/129.9 128.1/128.0 133.1/133.0

169.3, 21.4 3/7− 165.5/166.1 130.0/129.4 129.7/130.1 128.7/128.7 133.5/133.8

OAcAc 7− 165.4 129.1 130.2 128.7 133.8

17

δH, mult (Hz)

δH, mult (Hz)

α 3.01 m β 2.22 m 2.50 m 5.83 d (3.2) 2.97 m 5.63 d (8.0) 6.12 s 5.45 m 6.06 d (16.1) 5.96 dd (16.1, 9.0) 3.55 m 1.04 d (6.6) 5.51 s 5.47 m 1.21 s 1.32 s 1.44 d (6.6)

α 3.06 d (13.4, 6.9) β 1.63 t (13.2) 2.25 m 5.52 t (3.2) 2.58 d (3.1) 5.70 s 5.49 s 5.72 s 6.25 d (15.9) 5.63 dd (15.9, 9.6) 3.65 m 0.91 d (6.6) 5.23 s 5.20 s 1.12 s 1.23 s 1.18 d (6,7)

1.25 s 2.12 s 2.20 s −3/7 7.97 m/7.98 m 7.41 m/7.41 m 7.55 m/7.54 m

2.11 s 2.14 −7 7.99 7.42 7.56 −15 1.63 −3 2.12

d (7.1) t (7.7) t (7.4) s s

(4.40) nm; IR (KBr) νmax 3607, 2984, 1719, 1451, 1369, 1271, 1216, 1138, 1098, 1069, 1002, 950, 716 cm−1; 1H and 13C NMR (CDCl3) data, Tables 1 and 2; (+)-HRESIMS m/z 677.2523 [M + Na]+ (calcd for C35H42O12Na, 677.2568). Euphoglomeruphane B (2): white, amorphous powder; [α]22D +15 (c 0.2, MeOH); UV (MeOH) λmax(log ε) 228 (4.22) nm; IR (KBr) νmax 3502, 2986, 1717, 1451, 1370, 1272, 1221, 1103, 1068, 1025, 930, 712 cm−1; 1H and 13C NMR (CDCl3) data, Tables 1 and 2; (+)-HRESIMS m/z 635.2463 [M + Na]+ (calcd for C33H40O11Na, 635.2463). Euphoglomeruphane C (3): white, amorphous powder; [α]22D +72 (c 0.1, MeOH); UV (MeOH) λmax(log ε) 228 (4.29) nm; IR (KBr) νmax 3502, 2989, 1717, 1452, 1368, 1271, 1223, 1112, 1069, 1026, 959, 711 cm−1; 1H and 13C NMR (CDCl3) data, Tables 1 and 2; (+)-HRESIMS m/z 635.2463 [M + Na]+ (calcd for C33H40O11Na, 635.2463). Euphoglomeruphane D (4): colorless crystals (MeOH); mp 220− 222 °C; [α]22D +75 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 230 (4.5) nm; IR (KBr) νmax 3566, 2981, 1717, 1395, 1368, 1273, 1221, 1112, 1068, 1033, 953, 710 cm−1; 1H and 13C NMR (CDCl3) data, Tables 1 and 2; (+)-HRESIMS m/z 783.2989 [M + Na]+ (calcd for C42H48O13 Na, 783.2987). Euphoglomeruphane E (5): white, amorphous powder; [α]22D −21 (c 0.1, MeOH); UV (MeOH) λmax(log ε) 229 (4.22) nm; IR (KBr) νmax 3420, 2982, 1718, 1418, 1373, 1262, 1220, 1103, 1032, 963, 712 cm−1; 1H and 13C NMR (CDCl3) data, Tables 1 and 2; (+)-HRESIMS m/z 695.2635 [M + Na]+ (calcd for C35H44O13Na, 695.2674). Euphoglomeruphane F (6): white, amorphous powder; [α]22D +6 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 229 (4.62) nm; IR (KBr)

3− 170.1, 170.6 21.3, 21.5

a

ND: the signal was not detected.

afforded compounds 7 (20.0 mg), 8 (5.6 mg), and 16 (12.9 mg) as well as crystallized compound 21 (34.4 mg). Fr. 9 (5.4 g) was fractionated by CC (silica gel, 200−300 mesh, eluted with a gradient of CHCl3−acetone, from 100:1 to 0:1) to give 10 subfractions (9A−9J). Fraction 9F was separated by CC (Sephadex LH-20, 150 × 1.2 cm) to yield five subfractions (9F1− 9F5). Compounds 12 (2.0 mg) and 13 (2.8 mg) were separated from fraction 9F3 by eluting with a gradient solvent system (CH3CN− H2O, 48:52, semipreparative HPLC). Fraction 9H was purified by CC (Sephadex LH-20, 130 × 2 cm) to give nine subfractions (9H1− 9H9). Fraction 9H6 was subjected to semipreparative HPLC (CH3CN−H2O, 52:48) to afford compound 22 (4.0 mg). Euphoglomeruphane A (1): colorless crystals (MeOH); mp 209− 211 °C; [α]22D +31 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 228 I

DOI: 10.1021/acs.jnatprod.8b00507 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 8. Cytotoxicity of the Jatrophane Diterpenoids 1−17 against MCF-7 and MCF-7/ADR Cell Linesa IC50 (μM)

IC50 (μM)

compd

MCF-7

MCF-7/ADR

compd

MCF-7

MCF-7/ADR

1 2 3 4 5 6 7 8 9

>100 >100 >100 >100 >100 >100 >100 51.1 ± 2.3 >100

>100 >100 >100 >100 >100 >100 >100 39.3 ± 3.9 >100

10 11 12 13 14 15 16 17 DOX

>100 >100 50.2 ± 1.6 >100 >100 >100 >100 >100 0.7 ± 0.1

>100 >100 >100 >100 >100 >100 >100 >100 64.8 ± 2.1

a

MTT method was used for determination of the IC50 values for the compounds. Data were analyzed with GraphPad Prism 5.0 software and presented as the mean ± SD of three independent tests. νmax 3420, 2983, 1717, 1452, 1373, 1262, 1227, 1096, 1027, 962, 710 cm−1 ; 1 H and 13C NMR (CDCl3 ) data, Tables 2 and 3; (+)-HRESIMS m/z 715.2681 [M + Na]+ (calcd for C38H44O12Na, 715.2725). Euphoglomeruphane G (7): white, amorphous powder; [α]22D +119 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 228 (4.35) nm; IR (KBr) νmax3396, 2994, 1718, 1456, 1363, 1262, 1220, 1108, 1069, 1015, 931, 712 cm−1; 1H and 13C NMR (CDCl3) data, Tables 3 and 4; (+)-HRESIMS m/z 619.2495 [M + Na]+ (calcd for C33H40O10Na, 619.2514). Euphoglomeruphane H (8): white, amorphous powder; [α]22D +173 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 228 (4.25) nm; IR (KBr) νmax 3420, 2974, 1716, 1418, 1363, 1262, 1222, 1098, 1025, 935, 710 cm−1; 1H and 13C NMR (CDCl3) data, Tables 3 and 4; (+) HRESIMS m/z 619.2500 [M + Na]+ (calcd for C33H40O10Na,619.2514). Euphoglomeruphane I (9): white, amorphous powder; [α]22D +113 (c 0.1, MeOH); UV (MeOH) λmax(log ε) 229 (4.52) nm; IR (KBr) νmax 3446, 2987, 1717, 1456, 1373, 1263, 1221, 1094, 1069, 1026, 944, 710 cm−1; 1H and 13C NMR (CD3COCD3) data, Tables 3 and 4; (+)-HRESIMS m/z 713.2548 [M + Na]+ (calculated for C38H42O12Na, 713.2568). Euphoglomeruphane J (10): white, amorphous powder; [α]22D +46 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 229 (4.00) nm; IR (KBr) νmax 3384, 2981, 1717, 1455, 1263, 1107, 1054, 1016, 710 cm−1 ; 1 H and 13C NMR (CDCl3 ) data, Tables 3 and 4; (+)-HRESIMS m/z 697.2607 [M + Na] + (calculated for C38H42O11Na, 697.2619). Euphoglomeruphane K (11): white, amorphous powder; [α]22D +49 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 229 (4.49) nm; IR (KBr) νmax 3420, 2978, 1717, 1418, 1396, 1263, 1106, 1033, 712 cm−1 ; 1 H and 13C NMR (CDCl3 ) data, Tables 4 and 5; (+)-HRESIMS m/z 679.2710 [M + Na] + (calculated for C35H44O12Na, 679.2725). Euphoglomeruphane L (12): white, amorphous powder; [α]22D +47 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 228 (4.26) nm; IR (KBr) νmax 3446, 2998, 1718, 1457, 1373, 1264, 1229, 1149, 1108, 1032, 941, 712 cm−1; 1H and 13C NMR (CDCl3) data, Tables 4 and 5; (+)-HRESIMS m/z 721.2814 [M + Na]+ (calcd for C37H46O13Na, 721.2830). Euphoglomeruphane M (13): white, amorphous powder; [α]22D +45 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 228 (4.22) nm; IR (KBr) νmax 3447, 2997, 1717, 1457, 1373, 1264, 1225, 1183, 1145, 1108, 1032, 941, 712 cm−1; 1H and 13C NMR (CDCl3) data, Tables 5 and 6; (+)-HRESIMS m/z 721.2811 [M + Na]+ (calcd for C37H46O13Na, 721.2830). Euphoglomeruphane N (14): white, amorphous powder; [α]22D +107 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 227 (3.88) nm; IR (KBr) νmax 3446, 2978, 1717, 1456, 1363, 1264, 1224, 1104, 1033, 713 cm−1; 1H and 13C NMR (CDCl3) data, Tables 5 and 6; (+)-HRESIMS m/z 593.2345 [M + Na]+ (calcd for C31H38O10Na, 593.2357).

Euphoglomeruphane O (15): white, amorphous powder; [α]22D +22 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 230 (4.22) nm; IR (KBr) νmax 3420, 2981, 1717, 1456, 1373, 1262, 1225, 1094, 1067, 1027, 942, 708 cm−1; 1H and 13C NMR (CDCl3) data, Tables 5 and 6; (+)-HRESIMS m/z 739.2706 [M + Na]+ (calcd for C40H44O12Na, 739.2725). Euphoglomeruphane P (16): white, amorphous powder; [α]22D +169 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 229 (4.47) nm; IR (KBr) νmax 3421, 2997, 1717, 1456, 1373, 1324, 1263, 1227, 1099, 1070, 1032, 947, 712 cm−1; 1H and 13C NMR (CDCl3) data, Tables 6 and 7; (+)-HRESIMS m/z 739.2700 [M + Na]+ (calcd for C40H44O12Na, 739.2725). Euphoglomeruphane Q (17): white, amorphous powder; [α]22D −32 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 231 (4.19) nm; IR (KBr) νmax 3446, 2981, 1718, 1456, 1373, 1318, 1262, 1229, 1095, 1055, 1033, 1017, 950, 716 cm−1; 1H and 13C NMR (CDCl3) data, Tables 6 and 7; (+)-HRESIMS m/z 677.2549 [M + Na]+ (calcd for C35H42O12Na, 677.2568). X-ray Crystallography Analysis. Suitable crystals of 1, 4, and 21 were collected on a Bruker APEX-II CCD diffractometer equipped with Cu Kα radiation (λ = 1.541 78 Å). The structures and absolute configurations of 1, 4, and 21 were solved and determined with the ShelXT28 program using Intrinsic Phasing.29 Crystallographic data for 1, 4, and 21 have been deposited with the Cambridge Crystallographic Data Centre, deposition numbers CCDC 1838010 (1), 1887469 (4), and 1838014 (21). The crystallographic data of these compounds are available, free of charge, from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_ request/cif. Cells and Cell Culture. The MCF-7 cell line and its doxorubicin (DOX)-resistant subline MCF-7/ADR were cultured in RPMI-1640 medium (with 10% heat inactivated fetal bovine serum) and maintained at 37 °C under 5% CO2. DOX (1 μg/mL) was added to the MCF-7/ADR cell lines to measure the MDR effects, and the cells were cultured for 2 weeks. Cytotoxicity and MDR Reversal Assays. MTT assays were performed to measure the cytotoxicity and reversal effects of the isolated compounds. Euphoglomeruphane A−Q (1−17) were dissolved in DMSO for the assays. Briefly, cells (MCF-7 and MCF7/ADR) were seeded (at 5 × 103 cells per well) in 96-well flatbottomed microtiter plates during their logarithmic growth phase. In the MDR reversal assays, the cells were exposed to anticancer agents (DOX) with or without P-gp inhibitors for 48 h. In the assays, MCF7/ADR cells were cultured in the presence of DOX with serial dilutions of the jatrophane diterpenoids for 48 h, after which 10 μL of MTT dye (5 mg/mL) was added to each well and the plates were incubated for another 4 h in a 5% CO2 and 37 °C incubator. The medium was removed without disturbing the cells and the formazan product in the wells, and then 150 μL of DMSO was added to dissolve the MTT formazan crystals. The absorbance at 570 nm was measured according to the optical density with a microplate reader. J

DOI: 10.1021/acs.jnatprod.8b00507 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

The IC50 values were calculated from the dose−response curves using GraphPad Prism 5.0 software.

(4) Gunther, G.; Hohmann, J.; Vasas, A.; Mathe, I.; Dombi, G.; Jerkovich, G. Phytochemistry 1998, 47, 1309−1313. (5) Jakupovic, J.; Jeske, F.; Morgenstern, T.; Tsichritzis, F.; Marco, J. A.; Berendsohn, W. Phytochemistry 1998, 47, 1583−1600. (6) Hohmann, J.; Vasas, A.; Gunther, G.; Dombi, G.; Blazso, G.; Falkay, G.; Mathe, I.; Jerkovich, G. Phytochemistry 1999, 51, 673−677. (7) Shi, Q.; Su, X.; Kiyota, H. Chem. Rev. 2008, 108, 4295−4327. (8) Vasas, A.; Sulyok, E.; Redei, D.; Forgo, P.; Szabo, P.; Zupko, I.; Berenyi, A.; Molnar, J.; Hohmann, J. J. Nat. Prod. 2011, 74, 1453− 1461. (9) Figueroa-Gonzalez, G.; Jacobo-Herrera, N.; Zentella-Dehesa, A.; Pereda-Miranda, R. J. Nat. Prod. 2012, 75, 93−97. (10) Aljancic, I. S.; Pesic, M.; Milosavljevic, S. M.; Todorovic, N. M.; Jadranin, M.; Miosavljevic, G.; Povrenovic, D.; Bankovic, J.; Tanic, N.; Markovic, I. D.; Ruzdijic, S.; Vajs, V. E.; Tesevic, V. V. J. Nat. Prod. 2011, 74, 1613−1620. (11) Rawal, M. K.; Shokoohinia, Y.; Chianese, G.; Zolfaghari, B.; Appendino, G.; Taglialatela-Scafati, O.; Prasad, R.; Di Pietro, A. J. Nat. Prod. 2014, 77, 2700−2706. (12) Nim, S.; Monico, A.; Rawal, M. K.; Duarte, N.; Prasad, R.; Di Pietro, A.; Ferreira, M. U. Planta Med. 2016, 82, 1180−1185. (13) Esposito, M.; Nim, S.; Nothias, L.; Gallard, J.; Rawal, M. K.; Costa, J.; Roussi, F.; Prasad, R.; Di Pietro, A.; Paolini, J.; Litaudon, M. J. Nat. Prod. 2017, 80, 479−487. (14) Szabó, D.; Keyzer, H.; Kaiser, H. E.; Molnár, J. Anticancer Res. 2000, 20, 4261−4274. (15) Wang, B.; Zhao, B.; Chen, Z. S.; Pang, L. P.; Zhao, Y. D.; Guo, Q.; Zhang, X. H.; Liu, Y.; Liu, G. Y.; Hao-Zhang; Zhang, X. Y.; Ma, L. Y.; Liu, H. M. Eur. J. Med. Chem. 2018, 143, 1535−1542. (16) Binkhathlan, Z.; Lavasanifar, A. Curr. Cancer Drug Targets 2013, 13, 326−346. (17) Corea, G.; Fattorusso, E.; Lanzotti, V.; Motti, R.; Simon, P. N.; Dumontet, C.; Di Pietro, A. J. Med. Chem. 2004, 47, 988−992. (18) Pesic, M.; Bankovic, J.; Aljancic, I. S.; Todorovic, N. M.; Jadranin, M.; Vajs, V. E.; Tekvic, V. V.; Vuckovic, I.; Momcilovic, M.; Markovic, I. D.; Tanic, N.; Ruzdijic, S. Food Chem. Toxicol. 2011, 49, 3165−3173. (19) Jadranin, M.; Pesic, M.; Aljancic, I. S.; Milosavljevic, S. M.; Todorovic, N. M.; Podolski-Renic, A.; Bankovic, J.; Tanic, N.; Markovic, I.; Vajs, V. E.; Tesevic, V. V. Phytochemistry 2013, 86, 208− 217. (20) Huang, Y.; Aisa, H. A. Phytochem. Lett. 2010, 3, 176−180. (21) Huang, Y.; Aisa, H. A. Helv. Chim. Acta 2010, 93, 1156−1161. (22) Lu, D.; Liu, Y.; Aisa, H. A. Fitoterapia 2014, 92, 244−251. (23) Hu, R.; Gao, J.; Rozimamat, R.; Aisa, H. A. Eur. J. Med. Chem. 2018, 146, 157−170. (24) Esposito, M.; Nothias, L.; Nedev, H.; Gallard, J.; Leyssen, P.; Retailleau, P.; Costa, J.; Roussi, F.; Iorga, B. I.; Paolini, J.; Litaudon, M. J. Nat. Prod. 2016, 79, 2873−2882. (25) Redei, D.; Forgo, P.; Molnar, J.; Szabo, P.; Zorig, T.; Hohmann, J. Tetrahedron 2012, 68, 8403−8407. (26) Shokoohinia, Y.; Chianese, G.; Zolfaghari, B.; Sajjadi, S.; Appendino, G.; Taglialatela-Scafati, O. Fitoterapia 2011, 82, 317−322. (27) Hohmann, J.; Redei, D.; Forgo, P.; Molnar, J.; Dombi, G.; Zorig, T. J. Nat. Prod. 2003, 66, 976−979. (28) Sheldrick, G. M. Acta Crystallogr., Sect. C: Struct. Chem. 2015, 71, 3−8. (29) Sheldrick, G. M. Acta Crystallogr., Sect. A: Found. Adv. 2015, 71 (1), 3−8. (30) Nothias-Scaglia, L. F.; Pannecouque, C.; Renucci, F.; Delang, L.; Neyts, J.; Roussi, F.; Costa, J.; Leyssen, P.; Litaudon, M.; Paolini, J. J. Nat. Prod. 2015, 78, 1277−1283.

Table 9. DOX Resistance Reversal Activity of Diterpenoids 1−17 at 10.0 μM Concentration in MCF-7/ADR Cellsa compd

IC50/DOX (μM)

RF

compd

IC50/DOX (μM)

RF

1 2 3 4 5 6 7 8 9 10

9.6 ± 5.5 21.6 ± 8.0 13.3 ± 6.1 9.9 ± 3.3 20.6 ± 3.7 28.0 ± 4.6 14.2 ± 3.3 13.1 ± 9.6 7.8 ± 2.3 9.5 ± 4.0

6.8 3.0 4.9 6.5 3.1 2.3 4.6 5.0 8.3 6.8

11 12 13 14 15 16 17 controlb VRP

5.0 ± 0.8 5.3 ± 2.0 12.9 ± 3.7 25.3 ± 6.7 7.3 ± 1.5 8.0 ± 2.0 10.3 ± 0.6 64.8 ± 2.1 4.7 ± 0.7

12.9 12.3 5.0 2.6 8.9 8.1 6.3 1.0 13.7

a

The IC50 value was determined after exposure to a series of DOX concentrations with different compounds at 10 μM in MCF-7/ADR cells. Reversal fold (RF, fold-change in drug sensitivity) = (IC50 without inhibitor)/(IC50 with inhibitor). Data were analyzed with GraphPad Prism 5.0 software and presented as mean ± SD for three independent tests. b0.0.1% DMSO was added as solvent control.



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00507. 1D and 2D NMR spectra and (+)-HRESIMS data of compounds 1−17, 1H and 13C NMR (800 MHz, acetone-d6) data of 9, structures of the isolated terpenoids (1−22), and key HMBC and NOESY correlations of 1 (PDF) Crystallographic data (CIF) Crystallographic data (CIF) Crystallographic data (CIF)



AUTHOR INFORMATION

Corresponding Author

*Tel: +86-991-3835679. Fax: +86-991-3835679. E-mail: haji@ ms.xjb.ac.cn. ORCID

H. A. Aisa: 0000-0003-4652-6879 Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS The authors gratefully acknowledge the National Natural Science Foundation of China (No. 21572263), the Strategic Priority Research Program of CAS (No. XDA12020356), and Central Asia Drug Discovery and Development Centre of Chinese Academy of Science.



REFERENCES

(1) Fakunle, C. O.; Connolly, J. D.; Rycroft, D. S. J. Nat. Prod. 1989, 52, 279−283. (2) Hohmann, J.; Vasas, A.; Gunther, G.; Methe, I.; Evanics, F.; Dombi, G.; Jerkovich, G. J. Nat. Prod. 1997, 60, 331−335. (3) Appendino, G.; Jakupovic, S.; Tron, G. C.; Jakupovic, J.; Milon, V.; Ballero, M. J. Nat. Prod. 1998, 61, 749−756. K

DOI: 10.1021/acs.jnatprod.8b00507 J. Nat. Prod. XXXX, XXX, XXX−XXX